Subunit c of the F1F0 ATP synthase is a small transmembrane protein composed of two antiparallel transmembrane helices connected by a more polar loop. It plays a central role in oxidative phosphorylation, being involved in both the translocation of H+ through F0, and conformational coupling to the active sites for STP synthesis on F1. We have found that the purified protein folds normally in an aqueous-organic solvent mixture (4:4:1 CHCl3:MeOH:H20). We plan to determine the 3D structure of the protein at pHOs above and below the pKa of the Asp residue involved in H+ translocation using 3D and 4D heteronuclear NMR with doubly labeled protein. Since the structural study of membrane proteins has been limited in large part by the difficulty in obtaining crystals from detergent solubilized intrinsic membrane proteins suitable for X-ray diffraction studies, the development of convenient and reliable conditions for solution structural studies would be enormously helpful. A wide range of solvent mixtures and detergents will be screened to find optimal conditions for native folding and NMR analysis of membrane proteins. The screening will involve simple probes and the known structure of subunit c. The test for folding will be to insert a nitroxide spin label (PROXYL-maleimide at a unique Cys) in one helix of the protein, and an easily followed probe (15N single amino acid type) located in positions on the adjacent helix known to be nearby from the NMR structure. The distance between the paramagnetic center and the 15N probe will be calculated from the increase in the paramagnetic component of T1 and T2 of the 15N. The strength of the interaction between helices will be determined by T1( measurements as a function of spin lock field strength and temperature. A wide range of 1H (nondeuterated) solvents and detergents can, and will, be evaluated by this heteronuclear approach.